T cells are key players in the defense against infectious disease and cancer. Although much is known about T-cell development, selection, signaling, activation and apoptosis, many of the crucial events are not yet understood at a mechanistic level. The goal of the research proposed here is to provide new molecular and structural insights into signaling mechanisms as relevant for T-cell function. The proposed research is a continuation of a successful project that has lead in the past to a number of important structural and functional insights into the structural organization of the TCR/CD3 complex, intracellular and extracellular interactions of T-cell surface receptors and intracellular signaling mechanisms. The research proposed here has the goal to define protein interactions between invariant and clonotypic components of the TCR complex, as well as between TCR components and ligands outside and inside the plasma membrane. This will provide new insights into the mechanisms of TCR signal transduction. In addition we propose to study intracellular interactions crucial for T-cell signaling. It appears that some of the interactions within the components of the TCR complex are weak and transient. Thus, the planned research also includes the development and application of sensitive techniques that can identify and characterize such weak interactions with spatial resolution. The current proposal will pursue the following specific aims: 1. Characterize structures and interactions of invariant and clonotypic components of the T-cell receptor complex 2. Study structure and interactions of the cytosolic adaptor protein Nek and its potential function as a cellular switch in T cells. 3. Investigate the mode of interactions between the phosphatase calcineurin between nuclear factor of activated T-cells and mechanisms for inhibition of T-cell activation